Compositions and methods for treating heart failure

ABSTRACT

Certain substituted benzamide derivatives selectively modulate the cardiac sarcomere, for example by potentiating cardiac myosin, and are useful in the treatment of systolic heart failure including congestive heart failure.

CROSS-REFERENCE TO RELATED APPLICATONS

This application is a continuation of application Ser. No. 11/040,478,filed Jan. 20, 2005, now U.S. Pat. No. 7,053,094 which is a continuationof application Ser. No. 10/327,219, filed Dec. 20, 2002, (now U.S. Pat.No. 6,908,923, issued Jun. 21, 2005), which application claims thebenefit of provisional U.S. Application Ser. No. 60/343,088, filed Dec.21, 2001, all of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant Number 1R43 HL66647-01. Accordingly, the United States Government may havecertain rights in this invention.

FIELD OF THE INVENTION

The invention relates to substituted benzamide derivatives, particularlyto compounds that selectively modulate the cardiac sarcomere, andspecifically to compounds, pharmaceutical formulations and methods oftreatment for systolic heart failure, including congestive heartfailure.

BACKGROUND OF THE INVENTION

The Cardiac Sarcomere

The “sarcomere” is an elegantly organized cellular structure found incardiac and skeletal muscle made up of interdigitating thin and thickfilaments; it comprises nearly 60% of cardiac cell volume. The thickfilaments are composed of “myosin,” the protein responsible fortransducing chemical energy (ATP hydrolysis) into force and directedmovement. Myosin and its functionally related cousins are called motorproteins. The thin filaments are composed of a complex of proteins. Oneof these proteins, “actin” (a filamentous polymer) is the substrate uponwhich myosin pulls during force generation. Bound to actin are a set ofregulatory proteins, the “troponin complex” and “tropomyosin,” whichmake the actin-myosin interaction dependent on changes in intracellularCa²⁺ levels. With each heartbeat, Ca²⁺ levels rise and fall, initiatingcardiac muscle contraction and then cardiac muscle relaxation (Robbins Jand Leinwand L A. (1999) Molecular Basis of Cardiovascular Disease,Chapter 8. editor Chien, K. R., W. B. Saunders, Philadelphia). Each ofthe components of the sarcomere contributes to its contractile response.

Myosin is the most extensively studied of all the motor proteins. Of thethirteen distinct classes of myosin in human cells, the myosin-II classis responsible for contraction of skeletal, cardiac, and smooth muscle.This class of myosin is significantly different in amino acidcomposition and in overall structure from myosin in the other twelvedistinct classes (Goodson H V and Spudich J A. (1993) Proc. Natl. Acad.Sci. USA 90:659-663). Myosin-II consists of two globular head domainslinked together by a long alpha-helical coiled-coiled tail thatassembles with other myosin-IIs to form the core of the sarcomere'sthick filament. The globular heads have a catalytic domain where theactin binding and ATP functions of myosin take place. Once bound to anactin filament, the release of phosphate (cf. ATP to ADP) leads to achange in structural conformation of the catalytic domain that in turnalters the orientation of the light-chain binding lever arm domain thatextends from the globular head; this movement is termed the powerstroke.This change in orientation of the myosin head in relationship to actincauses the thick filament of which it is a part to move with respect tothe thin actin filament to which it is bound (Spudich J A. (2001) NatRev Mol Cell Biol. 2(5):387-92). Un-binding of the globular head fromthe actin filament (also Ca²⁺ modulated) coupled with return of thecatalytic domain and light chain to their startingconformation/orientation completes the contraction and relaxation cycle.

Mammalian heart muscle consists of two forms of cardiac myosin, alphaand beta, and they are well characterized (Robbins, supra). The betaform is the predominant form (>90 percent) in adult human cardiacmuscle. Both have been observed to be regulated in human heart failureconditions at both transcriptional and translational levels (Miyatasupra), with the alpha form being down-regulated in heart failure.

The sequences of all of the human skeletal, cardiac, and smooth musclemyosins have been determined. While the cardiac alpha and beta myosinsare very similar (93% identity), they are both considerably differentfrom human smooth muscle (42% identity) and more closely related toskeletal myosins (80% identity). Conveniently, cardiac muscle myosinsare incredibly conserved across mammalian species. For example, bothalpha and beta cardiac myosins are >96% conserved between humans andrats, and the available 250-residue sequence of porcine cardiac betamyosin is 100% conserved with the corresponding human cardiac betamyosin sequence. Such sequence conservation contributes to thepredictability of studying myosin based therapeutics in animal basedmodels of heart failure.

The components of the cardiac sarcomere present targets for thetreatment of heart failure, for example by increasing contractility orfacilitating complete relaxation to modulate systolic and diastolicfunction, respectively.

Heart Failure

Congestive heart failure (“CHF”) is not a specific disease, but rather aconstellation of signs and symptoms, all of which are caused by aninability of the heart to adequately respond to exertion by increasingcardiac output. The dominant pathophysiology associated with CHF issystolic dysfunction, an impairment of cardiac contractility (with aconsequent reduction in the amount of blood ejected with eachheartbeat). Systolic dysfunction with compensatory dilation of theventricular cavities results in the most common form of heart failure,“dilated cardiomyopathy,” which is often considered to be one in thesame as CHF. The counterpoint to systolic dysfunction is diastolicdysfunction, an impairment of the ability to fill the ventricles withblood, which can also result in heart failure even with preserved leftventricular function. Congestive heart failure is ultimately associatedwith improper function of the cardiac myocyte itself, involving adecrease in its ability to contract and relax.

Many of the same underlying conditions can give rise to systolic and/ordiastolic dysfunction, such as atherosclerosis, hypertension, viralinfection, valvular dysfunction, and genetic disorders. Patients withthese conditions typically present with the same classical symptoms:shortness of breath, edema and overwhelming fatigue. In approximatelyhalf of the patients with dilated cardiomyopathy, the cause of theirheart dysfunction is ischemic heart disease due to coronaryatherosclerosis. These patients have had either a single myocardialinfarction or multiple myocardial infarctions; here, the consequentscarring and remodeling results in the development of a dilated andhypocontractile heart. At times the causative agent cannot beidentified, so the disease is referred to as “idiopathic dilatedcardiomyopathy.” Irrespective of ischemic or other origin, patients withdilated cardiomyopathy share an abysmal prognosis, excessive morbidityand high mortality.

The prevalence of CHF has grown to epidemic proportions as thepopulation ages and as cardiologists have become more successful atreducing mortality from ischemic heart disease, the most common preludeto CHF. Roughly 4.6 million people in the United States have beendiagnosed with CHF; the incidence of such diagnosis is approaching 10per 1000 after 65 years of age. Hospitalization for CHF is usually theresult of inadequate outpatient-therapy. Hospital discharges for CHFrose from 377,000 (in 1979) to 957,000 (in 1997) making CHF the mostcommon discharge diagnosis in people age 65 and over. The five-yearmortality from CHF approaches 50% (Levy D. (2002) New Engl J Med.347(18):1442-4). Hence, while therapies for heart disease have greatlyimproved and life expectancies have extended over the last severalyears, new and better therapies continue to be sought, particularly forCHF.

“Acute” congestive heart failure (also known as acute “decompensated”heart failure) involves a precipitous drop in heart function resultingfrom a variety of causes. For example in a patient who already hascongestive heart failure, a new myocardial infarction, discontinuationof medications, and dietary indiscretions may all lead to accumulationof edema fluid and metabolic insufficiency even in the resting state. Atherapeutic agent that increases heart function during such an acuteepisode could assist in relieving this metabolic insufficiency andspeeding the removal of edema, facilitating the return to the morestable “compensated” congestive heart failure state. Patients with veryadvanced congestive heart failure particularly those at the end stage ofthe disease also could benefit from a therapeutic agent that increasesheart function, for example, for stabilization while waiting for a hearttransplant. Other potential benefits could be provided to patientscoming off a bypass pump, for example, by administration of an agentthat assists the stopped or slowed heart in resuming normal function.Patients who have diastolic dysfunction (insufficient relaxation of theheart muscle) could benefit from a therapeutic agent that modulatesrelaxation.

Therapeutic Active Agents

Inotropes are drugs that increase the contractile ability of the heart.As a group, all current inotropes have failed to meet the gold standardfor heart failure therapy, i.e., to prolong patient survival. Inaddition, current agents are poorly selective for cardiac tissue, inpart leading to recognized adverse effects that limit their use. Despitethis fact, intravenous inotropes continue to be widely used in acuteheart failure (e.g., to allow for reinstitution of oral medications orto bridge patients to heart transplantation) whereas in chronic heartfailure, orally given digoxin is used as an inotrope to relieve patientsymptoms, improve the quality of life, and reduce hospital admissions.

Given the limitations of current agents, new approaches are needed toimprove cardiac function in congestive heart failure. The most recentlyapproved short-term intravenous agent, milrinone, is now nearly fifteenyears old. The only available oral drug, digoxin, is over 200 hundredyears old. There remains a great need for agents that exploit newmechanisms of action and may have better outcomes in terms of relief ofsymptoms, safety, and patient mortality, both short-term and long-term.New agents with an improved therapeutic index over current agents willprovide a means to achieve these clinical outcomes. The selectivity ofagents directed at the cardiac sarcomere (for example, by targetingcardiac beta myosin) will be an important means to achieve this improvedtherapeutic index. The present invention provides such agents(particularly sarcomere activating agents) and methods for theiridentification and use.

SUMMARY OF THE INVENTION

The present invention provides compounds, pharmaceutical compositionsand methods for the treatment of heart disease including CHF,particularly systolic heart failure. The compositions are selectivemodulators of the cardiac sarcomere, for example potentiating cardiacmyosin.

In one aspect, the invention relates to Formula I:

wherein:

-   -   R¹ is alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl, and substituted heteroaryl;    -   R² is acetyl, acetylamino, acetylene, alkoxycarbonyl,        carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, nitro,        and trifluoromethyl; and    -   R³ is aryl, substituted aryl, heteroaryl, and substituted        heteroaryl;        including single stereoisomers, mixtures of stereoisomers, and        the pharmaceutically acceptable salts thereof. The compounds of        Formula I are useful as active agents in practice of the methods        of treatment and in manufacture of the pharmaceutical        formulations of the invention, and as intermediates in the        synthesis of such active agents.

In another aspect, the invention relates to compounds represented byFormula I, wherein:

-   -   R¹ is alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl and substituted heteroaryl;    -   R² is acetyl, acetylamino, acetylene, alkoxycarbonyl,        carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, and        trifluoromethyl; and    -   R³ is aryl, substituted aryl, heteroaryl, and substituted        heteroaryl;        or a single stereoisomer, or mixture of stereoisomers, or a        pharmaceutically acceptable salt thereof. Preferred in this        aspect are those compounds isomers and salts where:    -   R¹ is optionally substituted aryl or heteroaryl, preferably        phenyl, substituted phenyl or pyridinyl (especially        3-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl, 3-methylphenyl,        pyridin-2-yl, pyridin-3-yl or pyridin-4-yl);    -   R² is acetyl, acetylene, alkoxycarbonyl, cyano, halo,        heteroaryl, hydrogen or trifluoromethyl (especially acetylene,        cyano, ethoxycarbonyl, fluoro, hydrogen, oxazol-2-yl,        pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl);        and    -   R³ is phenyl, optionally substituted with lower alkoxy, lower        alkyl, halo, hydroxy, hydroxy-lower alkyl or nitro, or R³ is        benzodioxolyl, furanyl, indolyl, isoxazolyl, pyrazolyl,        pyridinyl or thiophenyl, optionally substituted with lower        alkyl, lower alkoxy, or halo (especially 3-hydroxyphenyl,        4-hydroxyphenyl, 4-fluorophenyl, isoxazol-5-yl, pyridin-3-yl,        pyridin-4-yl, thiophen-2-yl or 5-methyl-thiophen-2-yl).

In still another aspect, the invention relates to compounds representedby Formula I, wherein:

-   -   R¹ is alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl, or substituted heteroaryl;    -   R² is nitro; and    -   R³ is aryl, substituted aryl, heteroaryl or substituted        heteroaryl;        provided that:    -   when R¹ is pyridin-3-yl, R³ is not 3-bromophenyl,        3-fluorophenyl, 4-fluorophenyl, furan-2-yl, 4-methoxyphenyl or        1-methyl-1H-pyrazol-3-yl;    -   when R¹ is 4-hydroxyophenyl, R³ is not furan-2-yl;    -   when R¹ is 4-fluorophenyl, R³ is not furan-2-yl or pyridin-4-yl;        and    -   when R³ is pyridin-4-yl, R¹ is not phenyl, 3-chlorophenyl,        4-hydroxyphenyl or 3-methylphenyl;        or a single stereoisomer, or mixture of stereoisomers, or a        pharmaceutically acceptable salt thereof. Preferred in this        aspect are those compounds isomers and salts where:    -   R¹ is optionally substituted aryl or heteroaryl, preferably        phenyl, substituted phenyl or pyridinyl (especially        3-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl, 3-methylphenyl,        pyridin-2-yl or pyridin-3-yl); and    -   R³ is optionally substituted aryl or optionally substituted        heteroaryl (especially phenyl, 4-fluorophenyl, 4-cyanophenyl,        4-methoxyphenyl, 4-methylphenyl, isoxazol-5-yl, thiophene-2-yl        or 5-methyl-thiophene-2-yl).

Yet other aspects, the invention relates to a pharmaceutical formulationincluding a pharmaceutically acceptable excipient, and to a method oftreatment for heart disease, each entailing a therapeutically effectiveamount of a compound represented by Formula I, wherein:

-   -   R¹ is alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl or substituted heteroaryl;    -   R² is acetyl, acetylamino, acetylene, alkoxycarbonyl,        carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, nitro        or trifluoromethyl; and    -   R³ is aryl, substituted aryl, heteroaryl or substituted        heteroaryl;        or a single stereoisomer, or mixture of stereoisomers, or a        pharmaceutically acceptable salt thereof. Preferred in this        aspect are the above-described preferred compounds, isomers and        salts, and those where:    -   R¹ is substituted phenyl (especially 4-fluorophenyl) and R² is        nitro;    -   R¹ is heteroaryl (especially pyridin-3-yl) and R² is fluoro;    -   R¹ is heteroaryl (especially pyridin-3-yl) and R² is nitro,        particularly where R³ is phenyl, 4-fluorophenyl, 4-cyanophenyl,        4-methoxyphenyl or 4-methylphenyl.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to myosin (particularly myosinIII, a cardiac myosin or β cardiac myosin), for example compounds thatwill displace or compete with the binding of the compounds of theinvention. The methods comprise combining a labeled compound of theinvention, myosin, and at least one candidate agent and determining thebinding of the candidate agent to myosin.

In a further aspect, the invention provides methods of screening formodulators of the activity of myosin. The methods comprise combining acompound of the invention, myosin, and at least one candidate agent anddetermining the effect of the candidate agent on the activity of myosin.

Other aspects and embodiments will be apparent to those skilled in theart form the following detailed description.

DETAILED DESCRIPTION

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

Ac = acetyl Boc = t-butyloxy carbonyl c- = cyclo CBZ = carbobenzoxy =benzyloxycarbonyl DCM = dichloromethane = methylene chloride = CH₂Cl₂DIEA = N,N-diisopropylethylamine DMF = N,N-dimethylformamide DMSO =dimethyl sulfoxide Et = ethyl EYOAc = ethyl acetate EtOH = ethanol GC =gas chromatograghy h = hour Me = methyl min = minute mL = milliliter Ph= phenyl PyBroP = bromo-tris-pyrrolidinophosphonium hexafluorophosphatert = room temperature s- = secondary t- = tertiary TFA = trifluoroaceticacid THF = tetrahydrofuran TLC = thin layer chromatography

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns (e.g., substitutedalkyl includes optionally substituted cycloalkyl groups, which in turnare defined as including optionally substituted alkyl groups,potentially ad infinitum) that are sterically impractical, syntheticallynon-feasible and/or inherently unstable.

“Alkyl” is intended to include linear, branched, or cyclic hydrocarbonstructures and combinations thereof. Lower alkyl refers to alkyl groupsof from 1 to 5 carbon atoms. Examples of lower alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like.Preferred alkyl groups are those of C₂₀ or below. More preferred alkylgroups are those of C₁₃ or below. Still more preferred alkyl groups arethose of C₆ and below. Cycloalkyl is a subset of alkyl and includescyclic hydrocarbon groups of from 3 to 13 carbon atoms. Examples ofcycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbomyl,adamantyl and the like. In this application, alkyl refers to alkanyl,alkenyl and alkynyl residues; it is intended to includecyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene isanother subset of alkyl, referring to the same residues as alkyl, buthaving two points of attachment. Examples of alkylene include ethylene(—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), dimethylpropylene (—CH₂C(CH₃)₂CH₂—)and cyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When an alkyl residuehaving a specific number of carbons is named, all geometric isomershaving that number of carbons are intended to be encompassed; thus, forexample, “butyl” is meant to include n-butyl, sec-butyl, isobutyl andt-butyl; “propyl” includes n-propyl and isopropyl.

The term “alkoxy” or “alkoxyl” refers to the group —O-alkyl, preferablyincluding from 1 to 8 carbon atoms of a straight, branched, cyclicconfiguration and combinations thereof attached to the parent structurethrough an oxygen. Examples include methoxy, ethoxy, propoxy,isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxyrefers to groups containing one to four carbons.

The term “substituted alkoxy” refers to the group —O-(substitutedalkyl). One preferred substituted alkoxy group is “polyalkoxy” or—O-(optionally substituted alkylene)-(optionally substituted alkoxy),and includes groups such as —OCH₂CH₂OCH₃, and glycol ethers such aspolyethyleneglycol and —O(CH₂CH₂O)_(x)CH₃, where x is an integer ofabout 2-20, preferably about 2-10, and more preferably about 2-5.Another preferred substituted alkoxy group is hydroxyalkoxy or—OCH₂(CH₂)_(y)OH, where y is an integer of about 1-10, preferably about1-4.

“Acyl” refers to groups of from 1 to 10 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. “Lower-acyl” refers to groups containing1 to 4 carbons and “acyloxy” refers to the group O-acyl.

The term “amino” refers to the group —NH₂. The term “substituted amino”refers to the group —NHR or —NRR where each R is independently selectedfrom the group: optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted amino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl and sulfonyl, e.g.,diethylamino, methylsulfonylamino, furanyl-oxy-sulfonamino.

“Aryl” and “heteroaryl” mean a 5-, 6- or 7-membered aromatic orheteroaromatic ring containing 0-4 heteroatoms selected from O, N or S;a bicyclic 9- or 10-membered aromatic or heteroaromatic ring systemcontaining 0-4 (or more) heteroatoms selected from O, N or S; or atricyclic 12- to 14-membered aromatic or heteroaromatic ring systemcontaining 0-4 (or more) heteroatoms selected from O, N or S. Thearomatic 6- to 14-membered aromatic carbocyclic rings include, e.g.,phenyl, naphthalene, indane, tetralin, and fluorene and the 5- to10-membered aromatic heterocyclic rings include, e.g., imidazole,oxazole, isoxazole, oxadiazole, pyridine, indole, thiophene,benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline,quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.

“Aralkoxy” refers to the group —O-aralkyl. Similarly, “heteroaralkoxy”refers to the group —O-heteroaralkyl; “aryloxy” refers to —O-aryl; and“heteroaryloxy” refers to the group —O-heteroaryl.

“Aralkyl” refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples include benzyl,phenethyl, phenylvinyl, phenylallyl and the like. “Heteroaralkyl” refersto a residue in which a heteroaryl moiety is attached to the parentstructure via an alkyl residue. Examples include furanylmethyl,pyridinylmethyl, pyrimidinylethyl and the like.

“ATPase” refers to an enzyme that hydrolyzes ATP. ATPases includeproteins comprising molecular motors such as the myosins.

“Halogen” or “halo” refers to fluorine, chlorine, bromine or iodine.Fluorine, chlorine and bromine are preferred. Dihaloaryl, dihaloalkyl,trihaloaryl etc. refer to aryl and alkyl substituted with a plurality ofhalogens, but not necessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

“Heterocycle” means a cycloalkyl or aryl residue in which one to four ofthe carbons is replaced by a heteroatom such as oxygen, nitrogen orsulfur. Examples of heterocycles that fall within the scope of theinvention include imidazoline, pyrrolidine, pyrazole, pyrrole, indole,quinoline, isoquipoline, tetrahydroisoquinoline, benzofuran,benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl,when occurring as a substituent), tetrazole, morpholine, thiazole,pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline,isoxazole, oxadiazole, dioxane, tetrahydrofuran and the like.“N-heterocyclyl” refers to a nitrogen-containing heterocycle as asubstituent residue. The term heterocyclyl encompasses heteroaryl, whichis a subset of heterocyclyl. Examples of N-heterocyclyl residues include4-morpholinyl, 4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl,3-thiazolidinyl, piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examplesof substituted heterocyclyl include 4-methyl-1-piperazinyl and4-benzyl-1-piperidinyl.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. A 1:1 mixture of a pairof enantiomers is a “racemic” mixture. The term “(.±.)” is used todesignate a racemic mixture where appropriate. “Diastereoisomers” arestereoisomers that have at least two asymmetric atoms, but which are notmirror-images of each other. The absolute stereochemistry is specifiedaccording to the Cahn-lngold-Prelog R-S system. When a compound is apure enantiomer the stereochemistry at each chiral carbon may bespecified by either R or S. Resolved compounds whose absoluteconfiguration is unknown can be designated (+) or (−) depending on thedirection (dextro- or levorotatory) which they rotate plane polarizedlight at the wavelength of the sodium D line. Certain of the compoundsdescribed herein contain one or more asymmetric centers and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)- or(S)-. The present invention is meant to include all such possibleisomers, including racemic mixtures, optically pure forms andintermediate mixtures. Optically active (R)- and (S)- isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thisinvention and, which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable acidaddition salts can be formed with inorganic acids and organic acids.Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

“Substituted-” alkyl, aryl, heteroaryl and heterocyclyl referrespectively to alkyl, aryl, heteroaryl and heterocyclyl wherein one ormore (up to about 5, preferably up to about 3) hydrogen atoms arereplaced by a substituent independently selected from the group:optionally substituted alkyl (e.g., fluoroalkyl), optionally substitutedalkoxy, alkylenedioxy (e.g. methylenedioxy), optionally substitutedamino (e.g., alkylamino and dialkylamino), optionally substitutedamidino, optionally substituted aryl (e.g., phenyl), optionallysubstituted aralkyl (e.g., benzyl), optionally substituted aryloxy(e.g., phenoxy), optionally substituted aralkoxy (e.g., benzyloxy),carboxy (—COOH), carboalkoxy (ie., acyloxy or —OOCR), carboxyalkyl(i.e., esters or —COOR), carboxamido, aminocarbonyl,benzyloxycarbonylamino (CBZ-amino), cyano, carbonyl, halogen, hydroxy,optionally substituted heteroaryl, optionally substituted heteroaralkyl,optionally substituted heteroaryloxy, optionally substitutedheteroaralkoxy, nitro, sulfanyl, sulfinyl, sulfonyl, and thio.

The term “sulfanyl” refers to the groups: —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocyclyl).

The term “sulfinyl” refers to the groups: —S(O)—H, —S(O)-(optionallysubstituted alkyl), —S(O)-(optionally substituted amino),—S(O)-(optionally substituted aryl), —S(O)-(optionally substitutedheteroaryl), and —S(O)-(optionally substituted heterocyclyl).

The term “sulfonyl” refers to the groups: —S(O₂)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), —S(O₂)-(optionally substituted heterocyclyl),—S(O₂)-(optionally substituted alkoxy), —S(O₂)-optionally substitutedaryloxy), —S(O₂)-(optionally substituted heteroaryloxy), and—S(O₂)-(optionally substituted heterocyclyloxy).

The term “therapeutically effective amount” or “effective amount” refersto that amount of a compound of Formula I that is sufficient to effecttreatment, as defined below, when administered to a mammal in need ofsuch treatment. The therapeutically effective amount will vary dependingupon the subject and disease condition being treated, the weight and ageof the subject, the severity of the disease condition, the particularcompound of Formula I chosen, the dosing regimen to be followed, timingof administration, the manner of administration and the like, all ofwhich can readily be determined by one of ordinary skill in the art.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   -   a) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   b) inhibiting the disease, that is, slowing or arresting the        development of clinical symptoms; and/or    -   c) relieving the disease, that is, causing the regression of        clinical symptoms.

Compounds of the Present Invention

The present invention is directed to the compounds represented byFormula I, which are selective modulators of the cardiac sarcomere(e.g., by stimulating or otherwise potentiating the activity of cardiacmyosin), as follows:

wherein:

-   -   R¹ is selected from the group consisting of alkyl, substituted        alkyl, aryl, substituted aryl, heteroaryl, and substituted        heteroaryl;    -   R² is selected from the group: acetyl, acetylamino, acetylene,        alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl, hydrogen,        nitrile, nitro, and trifluoromethyl; and    -   R³ is selected from the group consisting of aryl, substituted        aryl, heteroaryl, and substituted heteroaryl;        including single stereoisomers, mixtures of stereoisomers, and        the pharmaceutically acceptable salts thereof. The compounds of        Formula I are useful as active agents in practice of the methods        of treatment and in manufacture of the pharmaceutical        formulations of the invention, and as intermediates in the        synthesis of such active agents.

The compounds falling within the foregoing genus and its subgenera areuseful as modulators of the cardiac sarcomere. Some of the compoundswere obtained from commercially available compound libraries. Theprovisos in the claims are meant to distinguish between subject matterthat is patentable as a composition of matter vs. subject matter thatcan be claimed based on applicants' recognition of itstherapeutic/pharmaceutical utility.

Nomenclature

The compounds of Formula I can be named and numbered (e.g., usingAutoNom version 2.1) as described below. For example, the compound ofFormula IA:

i.e., the compound according to Formula I where R¹ is pyridin-3-yl, R²is trifluoromethyl and R³ is 4-fluorophenyl, can be named4-fluoro-N-[3-(pyridin-3-yloxy)-5-trifluoromethyl-phenyl]-benzamide.

The compound of Formula IB:

i.e., the compound according to Formula I where R¹ is 4-fluorophenyl, R²is nitro and R³ is isoxalyl-5-yl, can be named isoxazole-5-carboxylicacid [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide.

Synthesis of the Compounds of Formula I

The compounds of the invention can be synthesized utilizing techniqueswell known in the art. See, for example, Kislitsin et al. (2000) J. Org.Chem. 65(25), 8430-8438; Shkinyova et al. (2000) Tetrahedron Letters41(25), 49734975; Shevelev et al. (1995) Mendeleev Commun. (1995), (4),157-8; Kamifuji et al. (1993) Jpn. Kokai Tokkyo Koho JP 05043556; andCui (2001) German Patent Appin. No. 2000-10010002, each of which isincorporated by reference.

Syntheses of the compounds of Formula I are illustrated below withreference to Reaction Scheme 1.

Synthetic Reaction Parameters

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure, generally within a temperature range from−10° C. to 110° C. Further, except as employed in the Examples or asotherwise specified, reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about −10° C. to about 110° C. over a period ofabout 1 to about 24 hours; reactions left to run overnight average aperiod of about 16 hours.

The terms “solvent”, “organic solvent” or “inert solvent” each mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

When desired, the (R)- and (S)-isomers may be resolved by methods knownto those skilled in the art, for example by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallisation; via formation of diastereoisomericderivatives which may be separated, for example, by crystallisation,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticoxidation or reduction, followed by separation of the modified andunmodified enantiomers; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support, such as silica witha bound chiral ligand or in the presence of a chiral solvent. Forexample, a compound of Formula I can be dissolved in a lower alkanol andplaced on a Chiralpak AD (205×20 mm) column (Chiral Technologies, Inc.)conditioned for 60 min at 70% EtOAc in Hexane. It will be appreciatedthat where the desired enantiomer is converted into another chemicalentity by one of the separation procedures described above, a furtherstep may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

Brief Description of Reaction Scheme

Reaction Scheme 1 illustrates synthesis of the compounds of Formula I,starting from a substituted nitro-benzene, which is condensed with an R¹alcohol, optionally derivatized to introduce the R² substituent, reducedto the corresponding amino-benzene and acidified to afford Formula I.

It will be appreciated by those skilled in the art that one or more ofthe reactants, steps and/or conditions described with reference toReaction Scheme 1 may require adjustment to accommodate varioussubstituents, e.g., at R¹ to R³.

Starting Materials

The nitro-benzenes of Formula 101 (e.g., 3-fluoro-5-iodo-nitro-benzeneand 3,5-difluoro-nitro-benzene), the R¹-alcohols of Formula 102 (e.g.,4-fluoro-phenol and pyridin-3-ol) and the like are commerciallyavailable, e.g., from Aldrich Chemical Company, Milwaukee, Wis. Otherreactants are likewise commercially available or may be readily preparedby those skilled in the art using commonly employed syntheticmethodology.

Preparation of Formula 103

Referring to Reaction Scheme 1, Step 1, a nitro-benzene of Formula101(where X is F or NO₂, and Y is acetyl, acetylamino, CF₃, C(O)CH₃,C(O)O-alkyl, CN, H, halo, NO₂, or the like) and an alkyl-, aryl- orheteroaryl-alcohol of Formula 102 are contacted in a solvent (e.g., DMF)in the presence of a base (e.g., K₂CO₃). The solution is stirred for1-96 hours at room temperature to 150° C. to afford the correspondingalkoxy-, aryloxy- or heteroaryloxy-nitro-benzene of Formula 103, whichis conventionally isolated and purified. The compound of Formula 103 canbe carried forward to Reaction Scheme 1, Step 2 or directly to ReactionScheme 1, Step 3.

Preparation of Formula 105

Referring to Reaction Scheme 1, Step 2, conversion of the alkoxy-,aryloxy- or heteroaryloxy-nitro-benzene of Formula 103 to thecorresponding R²-substituted nitro-benzene of Formula 105 takes placewith a variety of reactants (generically illustrated as Formula 104) andconditions depending on the nature of R², for example, as follows:

-   -   Where R² is acetylene, a compound of Formula 103, wherein Y is I        or Br, is contacted with palladium (e.g., PdOAc) and copper        (e.g., Cul) catalysts, a base (e.g., diethylamine) and        trimethylsilylacetylene of Formula 104, followed by removal of        the trimethylsilyl with a fluoride source (e.g.,        tetrabutylammonium fluoride “TBAF”). The reaction takes place        over 1-48 hours at room temperature to 150° C., or for 1-30 min.        with microwave irradiation, to afford the corresponding        acetylene-substituted nitro-benzene of Formula 105.        Alternatetively, this reaction can be run on a compound of        Formula I where R² is I or Br (e.g., where a compound of Formula        103 is carried forward directly to Reaction Scheme 1, Step 3).    -   Where R² is heteroaryl, a compound of Formula 103 wherein Y is I        or Br, is contacted with a heteroaryl organometalic (e.g., an        organozinc, an organoboronic acid, or an organotin) of Formula        104 in the presence of a palladium catalyst. The reaction takes        place over 1-48 hours at room temperature to 150° C., or for        1-30 min. with microwave irradiation, to afford the        corresponding heteroaryl-substituted nitro-benzene of        Formula 105. Alternatetively, this reaction can be run on a        compound of Formula I where R² is I or Br (e.g., where a        compound of Formula 103 is carried forward directly to Reaction        Scheme 1, Step 3).        The R²-substituted nitro-benzene of Formula 105 can be        conventionally isolated and purified, or carried forward without        isolation and purification.        Preparation of Formula 106

Referring to Reaction Scheme 1, Step 3, a nitro-benzene of Formula 105is reduced under typical conditions (e.g., Pd/C with H² NH₄COOH orZn/HCl or SnCl₂). The solution is stirred for 1-48 hours at roomtemperature to 150° C. to afford the corresponding amino-benzene ofFormula 106, which is conventionally isolated and purified.

Preparation of Formula I

Referring to Reaction Scheme 1, Step 4, an amino-benzene of Formula 106is contacted with an aryl- or heteroaryl-acid or acid chloride ofFormula 107 under standard amide coupling conditions that readilyassessable by those skilled in the art using commonly employed syntheticmethodology to afford the corresponding compound of Formula I, which isconventionally isolated and purified.

Compounds prepared by the above-described process of the invention canbe identified, e.g., by the presence of a detectable amount of Formula106 or 107. While it is well known that pharmaceuticals must meetpharmacopoeia standards before approval and/or marketing, and thatsynthetic reagents (such as 4-fluoro-benzoic acid) and precursors (suchas Formula 106) should not exceed the limits prescribed by pharmacopoeiastandards, final compounds prepared by a process of the presentinvention may have minor, but detectable, amounts of such materialspresent, for example at levels in the range of 95% purity with no singleimpurity greater than 1%. These levels can be detected, e.g., byemission spectroscopy. It is important to monitor the purity ofpharmaceutical compounds for the presence of such materials, whichpresence is additionally disclosed as a method of detecting use of asynthetic process of the invention.

Preferred Processes and Last Steps

A substituted amino-benzene compound of Formula 106 is condensed with anaryl- or heteroaryl-acid or acid chloride of Formula 107 to afford thecorresponding compound of Formula I.

A racemic mixture of isomers of a compound of Formula I is placed on achromatography column and separated into (R)- and (S)- enantiomers.

A compound of Formula I is contacted with a pharmaceutically acceptableacid to form the corresponding acid addition salt.

A pharmaceutically acceptable acid addition salt of Formula I iscontacted with a base to form the corresponding free base of Formula I.

Preferred Compounds

Preferred for the compounds, pharmaceutical formulations, methods ofmanufacture and use of the present invention are the followingcombinations and permutations of substituent groups of Formula I(sub-grouped, respectively, in increasing order of preference):

-   -   R¹ is optionally substituted aryl or heteroaryl.    -    Especially where optionally substituted aryl is phenyl or        substituted phenyl, and/or where heteroaryl is pyridinyl.        -   Particularly pyridin-2-yl or pyridin-3-yl.        -    Preferably, R¹ is pyridin-3-yl and R² is fluoro.        -   Particularly phenyl optionally substituted with lower alkyl,            halo or hydroxy.        -    Preferably 3-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl            and 3-methylphenyl.            -   More preferably 4-fluorophenyl.            -    Even more preferably, R¹ is 4-fluorophenyl and R² is                nitro.            -    Most preferably, R¹ is pyridin-2-yl, pyridin-3-yl or                4-fluorophenyl.    -   R² is acetyl, acetylene, an alkyl ester, cyano, halo, optionally        substituted heteroaryl, hydrogen, nitro (subject to the provisos        identified above and in the claims) or trifluoromethyl.    -    Especially where halo is fluoro; where the alkyl ester is        ethoxycarbonyl; and/or where heteroaryl is oxazolyl, pyrazinyl,        pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl, optionally        substituted with lower alkoxy or oxo.        -   Particularly where optionally substituted heteroaryl is            oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly            3-methoxy-2-oxo-2H-pyridin-1-yl), pyridin-2-yl,            pyrimidin-2-yl, tetrazol-2-yl (particularly            1-methoxymethoxy-1H-tetrazol-5-yl,            2-methoxymethoxy-2H-tetrazol-5-yl) or thiazol-2-yl.        -    Most preferably R² is acetylene, cyano, ethoxycarbonyl,            fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl,            pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.    -   R³ is optionally substituted aryl or optionally substituted        heteroaryl.    -    Especially when R³ is optionally substituted aryl, where aryl        is phenyl, optionally substituted with lower alkoxy, lower        alkyl, halo (particularly bromo, chloro or fluoro), hydroxy,        hydroxy-lower alkyl or nitro.        -   Particularly 3-bromophenyl, 4-chlorophenyl, 4-cyanophenyl,            3-fluorophenyl, 4-fluorophenyl, 3-hydroxyphenyl,            4-hydroxyethylphenyl, 4-hydroxyphenyl, 3-methylphenyl,            4-methoxyphenyl, 3,4,5-trimethoxyphenyl, or 4-nitrophenyl.    -    Especially when R³ is optionally substituted heteroaryl, where        heteroaryl is benzodioxolyl, furanyl, indolyl, isoxazolyl,        pyrazolyl, pyridinyl or thiophenyl, optionally substituted with        lower alkyl, lower alkoxy, or halo.        -   Particularly benzo[1,3]dioxol-5-yl, furan-2-yl,            5-fluoro-1H-indol-2-yl, 5-methoxy-1H-indol-2-yl,            6-methoxy-1H-indol-2-yl, isoxazol-5-yl,            1-methyl-1H-pyrazol-3-yl, 6-chloro-pyridin-3-yl,            5,6-dichloro-pyridin-3-yl, 6-methoxy-pyridin-3-yl,            pyridin-4-yl, thiophen-2-yl, 5-chlorothiophen-2-yl, or            5-methylthiophen-2-yl.    -    Especially where R³ is substituted aryl or optionally        substituted heteroaryl.        -   Particularly substituted phenyl or optionally substituted            isoxazolyl, pyridinyl, or thiophenyl.        -    Preferably 3-hydroxyphenyl, 4-hydroxyphenyl,            4-fluorophenyl, isoxazol-5-yl, pyridin-3-yl, pyridin-4-yl,            thiophen-2-yl or 5-methyl-thiophen-2-yl.            -   More preferably 4-fluorophenyl.            -    Most preferably where R¹ is pyridin-3-yl.        -   Particularly where R¹ is optionally substituted aryl or            heteroaryl.        -    Preferably where optionally substituted aryl is phenyl or            substituted phenyl, and/or where heteroaryl is pyridinyl.            -   More preferably pyridin-2-yl or pyridin-3-yl.            -   More preferably phenyl optionally substituted with lower                alkyl, halo or hydroxy.            -    Even more preferably 3-chlorophenyl, 4-fluorophenyl,                4-hydroxyphenyl and 3-methylphenyl.                -   Still more preferably 4-fluorophenyl.                -    Most preferably, R¹ is pyridin-2-yl, pyridin-3-yl                    or 4-fluorophenyl.        -   Particularly where R² is acetyl, acetylene, an alkyl ester,            cyano, halo, optionally substituted heteroaryl, hydrogen,            nitro or trifluoromethyl.        -    Preferably where halo is fluoro; where the alkyl ester is            ethoxycarbonyl; and/or where heteroaryl is oxazolyl,            pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl,            optionally substituted with lower alkoxy or oxo.            -   More preferably where optionally substituted heteroaryl                is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly                3-methoxy-2-oxo-2H-pyridin-1-yl), pyridin-2-yl,                pyrimidin-2-yl, tetrazol-2-yl (particularly                1-methoxymethoxy-1 H-tetrazol-5-yl,                2-methoxymethoxy-2H-tetrazol-5-yl) or thiazol-2-yl.            -    Most preferably R² is acetylene, cyano, ethoxycarbonyl,                fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl,                pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.    -    Especially where R¹ is optionally substituted aryl or        heteroaryl.        -   Particularly where optionally substituted aryl is phenyl or            substituted phenyl, and/or where heteroaryl is pyridinyl.        -    Preferably pyridin-2-yl or pyridin-3-yl.        -    Preferably phenyl optionally substituted with lower alkyl,            halo or hydroxy.            -   More preferably 3-chlorophenyl, 4-fluorophenyl,                4-hydroxyphenyl and 3-methylphenyl.            -    Even more preferably 4-fluorophenyl.                -   Most preferably, R¹ is pyridin-2-yl, pyridin-3-yl or                    4-fluorophenyl.        -   Particularly where R² is acetyl, acetylene, an alkyl ester,            cyano, halo, optionally substituted heteroaryl, hydrogen,            nitro or trifluoromethyl.        -    Preferably where halo is fluoro; where the alkyl ester is            ethoxycarbonyl; and/or where heteroaryl is oxazolyl,            pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl,            optionally substituted with lower alkoxy or oxo.            -   More preferably where optionally substituted heteroaryl                is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly                3-methoxy-2-oxo-2H-pyridin-1-yl), pyridin-2-yl,                pyrimidin-2-yl, tetrazol-2-yl (particularly                1-methoxymethoxy-1H-tetrazol-5-yl,                2-methoxymethoxy-2H-tetrazol-5-yl) or thiazol-2-yl.            -    Most preferably R² is acetylene, cyano, ethoxycarbonyl,                fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl,                pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.    -    Especially where R² is acetyl, acetylene, an alkyl ester,        cyano, halo, optionally substituted heteroaryl, hydrogen, nitro        or trifluoromethyl.        -   Particularly where halo is fluoro; where the alkyl ester is            ethoxycarbonyl; and/or where heteroaryl is oxazolyl,            pyrazinyl, pyridinyl, pyrimidihyl, tetrazolyl or thiazolyl,            optionally substituted with lower alkoxy or oxo.        -    Preferably where optionally substituted heteroaryl is            oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly            3-methoxy-2-oxo-2H-pyridin-1-yl), pyridin-2-yl,            pyrimidin-2-yl, tetrazol-2-yl (particularly            1-methoxymethoxy-1H-tetrazol-5-yl,            2-methoxymethoxy-2H-tetrazol-5-yl) or thiazol-2-yl.            -   More preferably R² is acetylene, cyano, ethoxycarbonyl,                fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl,                pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.    -   R¹ is heteroaryl and R³ is substituted phenyl.    -    Especially where R¹ is pyridin-3-yl.        -   Particularly where R² is acetylene, cyano, ethoxy-carbonyl,            fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl,            pyrazin-2-yl, thiazol-2-yl or trifluoromethyl.        -    Preferably where R² is nitro or fluoro.        -    Preferably where R³ is 4-fluorophenyl.        -   Particularly, as novel compositions of matter, where R² is            cyano, ethoxycarbonyl, hydrogen, fluoro, hydrogen,            pyridin-2-yl, pyrazin-2-yl, or trifluoromethyl.    -    Especially where R³ is 4-fluorophenyl.        -   Preferably where R¹ is pyridin-3-yl.    -   R¹ is substituted phenyl and R² is nitro.    -    Especially where R¹ is 4-fluorophenyl.    -   R¹ is heteroaryl and R² is fluoro.    -    Especially where R¹ is pyridin-3-yl.    -   R¹ is heteroaryl and R² is nitro.    -    Especially where R¹ is pyridin-3-yl.        -   Particularly where R³ is optionally substituted aryl.        -    Preferably where R³ is phenyl, 4-fluorophenyl,            4-cyanophenyl, 4-methoxyphenyl or 4-methylphenyl.            As illustrated with regard to the group of preferred            compounds where R³ is optionally substituted aryl,            optionally substituted heteroaryl or optionally substituted            heteroarylamino, the above-described groups and sub-groups            are individually preferred and can be combined to describe            further preferred aspects of the invention.

Particularly preferred (individually and collectively) for thepharmaceutical formulations, methods of manufacture and use of thepresent invention are the following:

-   4-fluoro-N-[nitro-5-(pyidin-3-yloxy)-phenyl]-benzamide;-   N-[3-(4-fluoro-phenoxy)-(5-nitro-phenyl]-isonicotinamide;-   4-fluoro-N-[3-(pyridin-3-yloxy)-phenyl]-benzamide;-   thiophene-2-carboxylic acid    [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide;-   isoxazole-5-carboxylic acid    [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide;-   N-[3-cyano-5-(pyridin-3-yloxy)-phenyl]]-4-fluoro-benzamide;-   4-fluoro-N-[3-(pyridin-3-yloxy)-5-trifluoromethyl-phenyl]-benzamide;-   3-(4-fluoro-benzoylamino)-5-(pyridin-3-yloxy)-benzoic acid ethyl    ester;-   5-methyl-thiophene-2-carboxylic acid    [3-nitro-5-(pyridin-3-yloxy)phenyl]-amide;-   4-fluoro-N-[3-pyridin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;-   4-fluoro-N-[3-pyrazin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;-   4-fluoro-N-[3-nitro-5-(pyridin-2-yloxy)-phenyl]-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-3-hydroxy-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-4-hydroxy-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-nicotinamide; and-   isoxazole-5-carboxylic acid    [3-fluoro-5-(pyridin-3-yloxy)-phenyl)-amide.

More preferred (individually and collectively) as novel compounds of thepresent invention, including their formulations, methods of manufactureand use, are the following:

-   4-fluoro-N-[3-(pyridin-3-yloxy)-phenyl]-benzamide;-   thiophene-2-carboxylic acid    [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide;-   isoxazole-5-carboxylic acid    [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide;-   N-[3-cyano-5-(pyridin-3-yloxy)-phenyl]]-4-fluoro-benzamide;-   4-fluoro-N-[3-(pyridin-3-yloxy)-5-trifluoromethyl-phenyl]-benzamide;-   3-(4-fluoro-benzoylamino)-5-(pyridin-3-yloxy)-benzoic acid ethyl    ester;-   5-methyl-thiophene-2-carboxylic acid    [3-nitro-5-(pyridin-3-yloxy)phenyl]-amide;-   4-fluoro-N-[3-pyridin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;-   4-fluoro-N-[3-pyrazin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;-   4-fluoro-N-[3-nitro-5-(pyridin-2-yloxy)-phenyl]-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-3-hydroxy-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]4-hydroxy-benzamide;-   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-nicotinamide; and-   isoxazole-5-carboxylic acid    [3-fluoro-5-(pyridin-3-yloxy)-phenyl)-amide.

Utility, Testing and Administration

Utility

The compounds of the present invention are selective for and modulatethe cardiac sarcomere, and are useful to bind to and/or potentiate theactivity of cardiac myosin, increasing the rate at which myosinhydrolyzes ATP. As used in this context, “modulate” means eitherincreasing or decreasing myosin activity, whereas “potentiate” means toincrease activity. It has also been determined in testing representativecompounds of the invention, that their administration can also increasethe contractile force in cardiac muscle fiber.

The compounds, pharmaceutical formulations and methods of the inventionare used to treat heart disease, including but not limited to: acute (ordecompensated) congestive heart failure, and chronic congestive heartfailure; particularly diseases associated with systolic heartdysfunction. Additional therapeutic utilities include administration tostabilize heart function in patients awaiting a heart transplant, and toassist a stopped or slowed heart in resuming normal function followinguse of a bypass pump.

Testing

ATP hydrolysis is employed by myosin in the sarcomere to produce force.Therefore, an increase in ATP hydrolysis would correspond to an increasein the force or velocity of muscle contraction. In the presence ofactin, myosin ATPase activity is stimulated >100 fold. Thus, ATPhydrolysis not only measures myosin enzymatic activity but also itsinteraction with the actin filament. A compound that modulates thecardiac sarcomere can be identified by an increase or decrease in therate of ATP hydrolysis by myosin, preferably exhibiting a 1.4 foldincrease at concentrations less than 10 μM (more preferably, less than 1μM). Preferred assays for such activity will employ myosin from a humansource, although myosin from other organisms can also be used. Systemsthat model the regulatory role of calcium in myosin binding are alsopreferred.

Alternatively, a biochemically functional sarcomere preparation can beused to determine in vitro ATPase activity, for example, as described inU.S. Ser. No. 09/539,164, filed Mar. 29, 2000. The functionalbiochemical behavior of the sarcomere, including calcium sensitivity ofATPase hydrolysis, can be reconstituted by combining its purifiedindividual components (particularly including its regulatory componentsand myosin). Another functional preparation is the in vitro motilityassay. It can be performed by adding test compound to a myosin-boundslide and observing the velocity of actin filaments sliding over themyosin covered glass surface (Kron S J. (1991) Methods Enzymol.196:399-416).

The in vitro rate of ATP hydrolysis correlates to myosin potentiatingactivity, which can be determined by monitoring the production of eitherADP or phosphate, for example as described in Ser. No. 09/314,464, filedMay 18, 1999. ADP production can also be monitored by coupling the ADPproduction to NADH oxidation (using the enzymes pyruvate kinase andlactate dehydrogenase) and monitoring the NADH level either byabsorbance or fluorescence (Greengard, P., Nature 178 (Part 4534):632-634 (1956); Mol Pharmacol 1970 Jan;6(1):31-40). Phosphate productioncan be monitored using purine nucleoside phosphorylase to couplephosphate production to the cleavage of a purine analog, which resultsin either a change in absorbance (Proc Natl Acad Sci U S A 1992 Jun1;89(11):4884-7) or fluorescence (Biochem J 1990 Mar 1;266(2):611-4).While a single measurement can be employed, it is preferred to takemultiple measurements of the same sample at different times in order todetermine the absolute rate of the protein activity; such measurementshave higher specificity particularly in the presence of test compoundsthat have similar absorbance or fluorescence properties with those ofthe enzymatic readout.

Test compounds can be assayed in a highly parallel fashion usingmultiwell plates by placing the compounds either individually in wellsor testing them in mixtures. Assay components including the targetprotein complex, coupling enzymes and substrates, and ATP can then beadded to the wells and the absorbance or fluorescence of each well ofthe plate can be measured with a plate reader.

A preferred method uses a 384 well plate format and a 25 μL reactionvolume. A pyruvate kinase/lactate dehydrogenase coupled enzyme system(Huang T G and Hackney D D. (1994) J Biol Chem 269(23):16493-16501) isused to measure the rate of ATP hydrolysis in each well. As will beappreciated by those in the art, the assay components are added inbuffers and reagents. Since the methods outlined herein allow kineticmeasurements, incubation periods are optimized to give adequatedetection signals over the background. The assay is done in real timegiving the kinetics of ATP hydrolysis, which increases the signal tonoise ratio of the assay.

Modulation of cardiac muscle fiber contractile force can be measuredusing detergent permeabilized cardiac fibers (also referred to asskinned cardiac fibers), for example, as described by Haikala H, et al(1995) J Cardiovasc Pharmacol 25(5):794-801. Skinned cardiac fibersretain their intrinsic sarcomeric organization, but do not retain allaspects of cellular calcium cycling, this model offers two advantages:first, the cellular membrane is not a barrier to compound penetration,and second, calcium concentration is controlled. Therefore, any increasein contractile force is a direct measure of the test compound's effecton sarcomeric proteins. Tension measurements are made by mounting oneend of the muscle fiber to a stationary post and the other end to atransducer that can measure force. After stretching the fiber to removeslack, the force transducer records increased tension as the fiberbegins to contract. This measurement is called the isometric tension,since the fiber is not allowed to shorten. Activation of thepermeabilized muscle fiber is accomplished by placing it in a bufferedcalcium solution, followed by addition of test compound or control. Whentested in this manner, compounds of the invention caused an increase inforce at calcium concentrations associated with physiologic contractileactivity, but very little augmentation of force in relaxing buffer atlow calcium concentrations or in the absence of calcium (the EGTA datapoint).

Selectivity for the cardiac sarcomere and cardiac myosin can bedetermined by substituting non-cardiac sarcomere components and myosinin one or more of the above-described assays and comparing the resultsobtained against those obtained using the cardiac equivalents.

Initial evaluation of in vivo activity can be determined in cellularmodels of myocyte contractility, e.g., as described by Popping S, et al((1996) Am. J. Physiol. 271: H357-H364) and Wolska B M, et al ((1996)Am. J. Physiol. 39:H24-H32). One advantage of the myocyte model is thatthe component systems that result in changes in contractility can beisolated and the major site(s) of action determined. Compounds withcellular activity (for example, selecting compounds having the followingprofile: >120% increase in fractional shortening over basal at 2 μM,limited changes in diastolic length (<5% change), and no significantdecrease in contraction or relaxation velocities) can then be assessedin whole organ models, such as such as the Isolated Heart (Langendorff)model of cardiac function, in vivo using echocardiography or invasivehemodynamic measures, and in animal-based heart failure models, such asthe Rat Left Coronary Artery Occlusion model. Ultimately, activity fortreating heart disease is demonstrated in blinded, placebo-controlled,human clinical trials.

Administration

The compounds of Formula I are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. While human dosage levels have yetto be optimized for the compounds of the invention, generally, a dailydose is from about 0.05 to 100 mg/kg of body weight, preferably about0.10 to 10.0 mg/kg of body weight, and most preferably about 0.15 to 1.0mg/kg of body weight. Thus, for administration to a 70 kg person, thedosage range would be about 3.5 to 7000 mg per day, preferably about 7.0to 700.0 mg per day, and most preferably about 10.0 to 100.0 mg per day.The amount of active compound administered will, of course, be dependenton the subject and disease state being treated, the severity of theaffliction, the manner and schedule of administration and the judgmentof the prescribing physician; for example, a likely dose range for oraladministration would be about 70 to 700 mg per day, whereas forintravenous administration a likely dose range would be about 700 to7000 mg per day, the active agents being selected for longer or shorterplasma half-lives, respectively.

Administration of the compounds of the invention or the pharmaceuticallyacceptable salts thereof can be via any of the accepted modes ofadministration for agents that serve similar utilities including, butnot limited to, orally, subcutaneously, intravenously, intranasally,topically, transdermally, intraperitoneally, intramuscularly,intrapulmonarilly, vaginally, rectally, or intraocularly. Oral andparenteral administration are customary in treating the indications thatare the subject of the present invention.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, e.g., tablets, capsules,powders, liquids, suspensions, suppositories, aerosols or the like. Thecompounds can also be administered in sustained or controlled releasedosage forms, including depot injections, osmotic pumps, pills,transdermal (including electrotransport) patches, and the like, forprolonged and/or timed, pulsed administration at a predetermined rate.Preferably, the compositions are provided in unit dosage forms suitablefor single administration of a precise dose.

The compounds can be administered either alone or more typically incombination with a conventional pharmaceutical carrier, excipient or thelike (e.g., mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like). If desired, thepharmaceutical composition can also contain minor amounts of nontoxicauxiliary substances such as wetting agents, emulsifying agents,solubilizing agents, pH buffering agents and the like (e.g., sodiumacetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine acetate, triethanolamine oleate, and thelike). Generally, depending on the intended mode of administration, thepharmaceutical formulation will contain about 0.005% to 95%, preferablyabout 0.5% to 50% by weight of a compound of the invention. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

In addition, the compounds of the invention can be co-administered with,and the pharmaceutical compositions can include, other medicinal agents,pharmaceutical agents, adjuvants, and the like. Suitable additionalactive agents include, for example: therapies that retard theprogression of heart failure by down-regulating neurohormonalstimulation of the heart and attempt to prevent cardiac remodeling(e.g., ACE inhibitors or β-blockers); therapies that improve cardiacfunction by stimulating cardiac contractility (e.g., positive inotropicagents, such as the β-adrenergic agonist dobutamine or thephosphodiesterase irihibitor milrinone); and therapies that reducecardiac preload (e.g., diuretics, such as furosemide).

In one preferred embodiment, the compositions will take the form of apill or tablet and thus the composition will contain, along with theactive ingredient, a diluent such as lactose, sucrose, dicalciumphosphate, or the like; a lubricant such as magnesium stearate or thelike; and a binder such as starch, gum acacia, polyvinylpyrrolidine,gelatin, cellulose, cellulose derivatives or the like. In another soliddosage form, a powder, marume, solution or suspension (e.g., inpropylene carbonate, vegetable oils or triglycerides) is encapsulated ina gelatin capsule.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, asemulsions, or in solid forms suitable for dissolution or suspension inliquid prior to injection. The percentage of active compound containedin such parenteral compositions is highly dependent on the specificnature thereof, as well as the activity of the compound and the needs ofthe subject. However, percentages of active ingredient of 0.01% to 10%in solution are employable, and will be higher if the composition is asolid which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, preferably less than10 microns.

Use in Screening

Generally, to employ the compounds of the invention in a method ofscreening for myosin binding, myosin is bound to a support and acompound of the invention is added to the assay. Alternatively, thecompound of the invention can be bound to the support and the myosinadded. Classes of compounds among which novel binding agents may besought include specific antibodies, non-natural binding agentsidentified in screens of chemical libraries, peptide analogs, etc. Ofparticular interest are screening assays for candidate agents that havea low toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like. See,e.g., U.S. Pat. No. 6,495,337, incorporated herein by reference.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 N-[3-Cyano-5-(pyridin-3-yloxy)-phenyl]]4-fluoro-benzamide

1A. Formula 103 where R¹ is Pyridin-3-yl and Y is CN

A solution of 1 equivalent (eq., 25.42 g, 131.6 mmol)) of 1,3dinitro-5-cyanobenzene in 1.75 M DMF (75 mL), 1 eq. 3-hydroxypyridine(12.52 g, 131.6 mmol) and 2 eq. potassium carbonate (36.38 g, 263 mmol)was heated to 60° C. overnight. The reaction mixture was warmed to 95°C. for an additional 48 hours. The reaction mixture was diluted withEtOAc, washed with H₂O, sat. NaHCO₃, and brine, dried over Na₂SO₄ andconcentrated in vacuo. Purification by chromatography over silica with25% EtOAC/Hexane as the eluant afforded 10.04 g of the desired compoundof Formula 103, 3-(pyridin-3-yloxy)-5-cyano-1-nitrobenzene, as a solid(32% yield).

1B. Formula 106 where R¹ is Pyridin-3-yl and R² is CN

To a solution of 1 eq. (5.5 g, 22.8 mmol) of3-(pyridin-3-yloxy)-5-cyano-1-nitrobenzene 0.23 M in EtOH (100 mL) wasadded 3 eq. of tin (II) chloride (15.5 g, 68 mmol) and the resultantmixture stirred under a nitrogen atmosphere at rt overnight. Thereaction mixture was diluted with water (100 mL) and the pH adjusted to9 by the addition of saturated Na₂CO₃. The mixture was extracted withEtOAc (3 times), the combined organic layers dried (Na₂SO₄) and conc. invacuo. Purification by chromatography over silica with 2% MeOH/CH₂Cl₂ asthe eluant afforded 1.09 g of the desired compound of Formula 103,3-(pyridin-3-yloxy)-5-cyano-aniline, as a solid (23% yield).

1C. Formula I where R¹ is Pyridin-3-yl R² is CN, and R³ is4-Fluorophenyl

To a solution of 1 eq. (0.1 g, 0.473 mmol) of3-(pyridin-3-yloxy)-5-cyano-aniline in 0.47 M in CH₂Cl₂ (1 mL) and 1.6eq. of pyridine (0.56 g, 0.71 mmol) was added 3 eq. of 4-fluorobenzoylchloride (0.225 g, 1.42 mmol) and the resultant mixture stirred at rtovernight. The reaction mixture was diluted with CH₂Cl₂, washed withsat. NaHCO₃, dried (Na₂SO₄) and concentrated in vacuo. The resultantresidue was diluted with toluene and concentrated in vacuo (5 times).Purification by chromatography over silica with 5% MeOH/CH₂Cl₂ as theeluant afforded 134 mg of a the desired title compound of Formula I,N-[3-cyano-5-(pyridin-3-yloxy)-phenyl]]-4-fluoro-benzamide, as a solid(85% yield). Mpt 169-172° C. MS (M−1) 332.3.

Example 24-Fluoro-N-[3-pyridin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide

2A. Formula I where R¹ is Pyridin-3-yl R² is Pyridin-2-yl- and R³ is4-Fluorophenyl

A mixture of 1 eq. (175 mg, 0.402 mmol) of4-fluoro-N-[3-iodo-5-(pyridin-3-yloxy)-phenyl]-benzamide 1.15 eq. (170mg, 0.463 mmol) of 2-tributyltinpyridine, and 0.04 eq. (11 mg, 0.10mmol) of bis-(triphenylphosphine)dichloropalladium in 2 mL of THF wasirradiated for in a microwave for 10 minutes an 170° C. The reactionmixture was filtered and concentrated in vacuo. Purification bychromatography over silica with 50% EtOAC/Hexane to 75% EtOAc/Hexane asthe gradient eluant afforded 30 mg of a solid(4-fluoro-N-[3-pyridin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide, (19%yield). Mpt 160-162° C. MS (M+1) 386.1.

Example 3 Other Compounds of Formula I

Similarly, by following the procedures of Examples 1 and/or 2, and e.g.,substituting the 1,3 dinitro-5-cyanobenzene, 3-hydroxypyridine,4-fluorobenzoyl chloride and/or 2-tributyltinpyridine as described inconnection with Reaction Scheme 1, there were obtained:

-   -   4-fluoro-N-[nitro-5-(pyidin-3-yloxy)-phenyl]-benzamide, MS (M+1        ) 354.2;    -   N-[3-(4-fluoro-phenoxy)-(5-nitro-phenyl]-isonicotinamide, MS        (M−1) 352.3;    -   4-fluoro-N-[3-(pyridin-3-yloxy)-phenyl]-benzamide, MS (M+1)        309.4;    -   thiophene-2-carboxylic acid        [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide, MS (M−1) 356.6;    -   isoxazole-5-carboxylic acid        [3-(4-fluoro-phenoxy)-5-nitro-phenyl-amide, MS (M−1) 341.6;    -   4-fluoro-N-[3-(pyridin-3-yloxy)-5-trifluoromethyl-phenyl]-benzamide,        MS (M+1) 377.2;    -   3-(4-fluoro-benzoylamino)-5-(pyridin-3-yloxy)-benzoic acid ethyl        ester, MS (M+1) 381.2;    -   5-methyl-thiophene-2-carboxylic acid        [3-nitro-5-(pyridin-3-yloxy)phenyl]-amide, MS (M−1) 353.9;    -   4-fluoro-N-[3-pyrazin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide,        MS (M−1) 385.4;    -   4-fluoro-N-[3-nitro-5-(pyridin-2-yloxy)-phenyl]-benzamide, MS        (M+1) 354.2;    -   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-3-hydroxy-benzamide, MS        (M+1) 325.1;    -   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]4-hydroxy-benzamide, MS        (M+1) 325.2;    -   N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-nicotinamide, MS (M+1)        310.1;    -   isoxazole-5-carboxylic acid        [3-fluoro-5-(pyridin-3-yloxy)-phenyl)-amide, MS (M+1) 300.1;    -   5,6-dichloro-N-[3-nitro-5-(pyridin-3-yloxy)-phenyl]-nicotinamide,        MS (M−1) 402.3;    -   4-fluoro-N-[3-[1-(2-methoxy-ethoxymethyl)-1H-tetrazol-5-yl]-5-(pyridin-3-yloxy)-phenyl]-benzamide,        MS (M+1) 465.3;    -   3-(4-fluoro-benzoylamino)-5-(pyridin-3-yloxy)-benzenesulfonic        acid pyridin-3-yl ester, MS (M+1) 466.1;    -   6-methoxy-1H-indole-2-carboxylic acid        [3-fluoro-5-(pyridin-3-yloxy)-phenyl]-amide, MS (M+1) 378.1; and    -   1-[3-(3-methoxy-2-oxo-2H-pyridin-1-yl)-5-(pyridin-3-yloxy)-phenyl]-3-(6-methoxy-pyridin        -3-yl)-urea, MS (M+1) 459.2.

Example 4 In vito Model of Dose Dependent Cardiac Myosin ATPaseModulation

Dose responses are measured using a calcium-buffered, pyruvate kinaseand lactate dehydrogenase-coupled ATPase assay containing the followingreagents (concentrations expressed are final assay concentrations):Potassium PIPES (12 mM), MgCl₂ (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactatedehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to6.80 at 22° C. by addition of potassium hydroxide. Calcium levels arecontrolled by a buffering system containing 0.6 mM EGTA and varyingconcentrations of calcium, to achieve a free calcium concentration of1×10⁻⁴ M to 1×10⁻⁸ M.

The protein components specific to this assay are bovine cardiac myosinsubfragment-1 (typically 0.5 μM), bovine cardiac actin (14 μM), bovinecardiac tropomyosin (typically 3 μM), and bovine cardiac troponin(typically 3-8 μM). The exact concentrations of tropomyosin and troponinare determined empirically, by titration to achieve maximal differencein ATPase activity when measured in the presence of 1 mM EGTA versusthat measured in the presence of 0.2 mM CaCl₂. The exact concentrationof myosin in the assay is also determined empirically, by titration toachieve a desired rate of ATP hydrolysis. This varies between proteinpreparations, due to variations in the fraction of active molecules ineach preparation.

Compound dose responses are typically measured at the calciumconcentration corresponding to 50% of maximal ATPase activity (pCa₅₀),so a preliminary experiment is performed to test the response of theATPase activity to free calcium concentrations in the range of 1×10⁻⁴ Mto 1×10⁻⁸ M. Subsequently, the assay mixture is adjusted to the pCas₅₀(typically 3×10⁻⁷ M). Assays are performed by first preparing a dilutionseries of test compound, each with an assay mixture containing potassiumPipes, MgCl₂, BSA, DTT, pyruvate kinase, lactate dehydrogenase, myosinsubfragment-1, antifoam, EGTA, CaCl₂, and water. The assay is started byadding an equal volume of solution containing potassium Pipes, MgCl₂,BSA, DTT, ATP, NADH, PEP, actin, tropomyosin, troponin, antifoam, andwater. ATP hydrolysis is monitored by absorbance at 340 nm. Theresulting dose response curve is fit by the 4 parameter equationy=Bottom+((Top-Bottom)/(1+((EC50/X)^Hill))). The AC1.4 is defined as theconcentration at which ATPase activity is 1.4-fold higher than thebottom of the dose curve.

Preferred compounds of the invention have an AC1.4 less than 10 μM; andmore preferably, less than 1 μM.

When tested as described above, compounds of Formula I show activity aspotentiators of cardiac myosin.

Example 5 Myocyte Calcium-Contractility Assay

5A. Preparations of Adult Cardiac Ventricular Rat Myocytes

Adult male Sprague-Dawley rats are anesthetized with a mixture ofisoflurane gas and oxygen. Hearts are quickly excised, rinsed and theascending aorta cannulated. Continuous retrograde perfusion is initiatedon the hearts at a perfusion pressure of 60 cm H₂O. Hearts are firstperfused with a nominally Ca²⁺ free modified Krebs solution of thefollowing composition: 110 mM NaCl, 2.6 mM KCL, 1.2 mM KH₂PO₄ 7 H₂O, 1.2mM MgSO₄, 2.1 mM NaHCO₃, 11 mM glucose and 4 mM Hepes (all Sigma). Thismedium is not recirculated and is continually gassed with O₂. Afterapproximately 3 minutes the heart is perfused with modified Krebs buffersupplemented with 3.3% collagenase (169 μ/mg activity, Class II,Worthington Biochemical Corp., Freehold, N.J.) and 25 μM final calciumconcentration until the heart becomes sufficiently blanched and soft.The heart is removed from the cannulae, the atria and vessels discardedand the ventricles are cut into small pieces. The myocytes are dispersedby gentle agitation of the ventricular tissue in fresh collagenasecontaining Krebs prior to being gently forced through a 200 μm nylonmesh in a 50 cc tube. The resulting myocytes are resuspended in modifiedKrebs solution containing 25 μm calcium. Myocytes are made calciumtolerant by addition of a calcium solution (100 mM stock) at 10 minuteintervals until 100 μM calcium is achieved. After 30 minutes thesupernatant is discarded and 30-50 ml of Tyrode buffer (137 mM NaCL, 3.7mM KCL, 0.5 mM MgCL, 11 mM glucose, 4 mM Hepes, and 1.2 mM CaCl₂, pH7.4) is added to cells. Cells are kept for 60 min at 37° C. prior toinitiating experiments and used within 5 hrs of isolation.

5B. Adult Ventricular Myocyte Contractility Experiments

Aliquots of Tyrode buffer containing myocytes are placed in perfusionchambers (series 20 RC-27NE; Warner Instruments) complete with heatingplatforms. Myocytes are allowed to attach, the chambers heated to 37°C., and the cells then perfused with 37° C. Tyrode buffer. Myocytes arefield stimulated at 1 Hz in with platinum electrodes (20% abovethreshold). Only cells that have clear striations, and are quiescentprior to pacing are used for contractility experiments. To determinebasal contractility, myocytes are imaged through a 40× objective andusing a variable frame rate (60-240 Hz) charge-coupled device camera,the images are digitized and displayed on a computer screen at asampling speed of 240 Hz. [Frame grabber, myopacer, acquisition, andanalysis software for cell contractility are available from IonOptix(Milton, Ma.).] After a minimum 5 minute basal contractility period,test compounds (0.01-15 μM) are perfused on the myocytes for 5 minutes.After this time, fresh Tyrode buffer is perfused to determine compoundwashout characteristics. Using edge detection strategy, contractility ofthe myocytes and contraction and relaxation velocities are continuouslyrecorded.

5C. Contractility Analysis

Three or more individual myocytes are tested per compound, using two ormore different myocyte preparations. For each cell, ten or morecontractility transients at basal (defined as 1 min. prior to compoundinfusion) and at 5 min. after compound addition, are averaged andcompared. These average transients are analyzed to determine changes indiastolic length, and using the lonwizard analysis program (IonOptix),fractional shortening (% decrease in the diastolic length), and maximumcontraction and relaxation velocities (μm/sec) are determined. Analysisof individual cells are combined. Increase in fractional shortening overbasal indicates potentiation of myocyte contractility.

5D. Results

Compounds of the present invention show activity when tested by thismethod.

Example 6 In vivo Fractional Shortening Assay

6A. Animals

Male Sprague Dawley rats from Charles River Laboratories (275-350 g) areused for bolus efficacy and infusion studies. Heart failure animals aredescribed below. They are housed two per cage and have access to foodand water ad libitum. There is a minimum three-day acclimation periodprior to experiments.

6B. Echocardiography

Animals are anesthetized with isoflurane and maintained within asurgical plane throughout the procedure. Core body temperature ismaintained at 37° C. by using a heating pad. Once anesthetized, animalsare shaven and hair remover is applied to remove all traces of fur fromthe chest area. The chest area is further prepped with 70% ETOH andultrasound gel is applied. Using a GE System Vingmed ultrasound system(General Electric Medical Systems), a 10 MHz probe is placed on thechest wall and images are acquired in the short axis view at the levelof the papillary muscles. 2-D M-mode images of the left ventricle aretaken prior to, and after, compound bolus injection or infusion. In vivofractional shortening ((end diastolic diameter−end systolic diameter)/end diastolic diameter ×100) is determined by analysis of the M-modeimages using the GE EchoPak software program.

6C. Bolus and Infusion Efficacy

For bolus injection, rats are treated as described above. Five pre-doseM-Mode images are taken at 30 second intervals prior to bolus injectionor infusion of compounds. After injection, M-mode images are taken at 30second intervals up to 10 minutes and every minute or at five minuteintervals thereafter. Bolus injection or infusion is via the tail vein.Infusion parameters are determined from pharmacokinetic profiles ofspecific compounds.

6D. Results

Compounds of the present invention show activity when tested by thismethod.

Example 7 Left Coronary Artery Occlusion Model of Congestive HeartFailure

7A. Animals

Male Sprague-Dawley CD (220-225 g; Charles River) rats are used in thisexperiment. Animals are allowed free access to water and commercialrodent diet under standard laboratory conditions. Room temperature ismaintained at 20-23° C. and room illumination is on a 12/12-hourlight/dark cycle. Animals are acclimatized to the laboratory environment5 to 7 days prior to the study. The animals are fasted overnight priorto surgery.

7B. Occlusion Procedure

Animals are anaesthetized with ketamine/xylazine (95 mg/kg and 5 mg/kg)and intubated with a 14-16-gauge modified intravenous catheter.Anesthesia level is checked by toe pinch. Core body temperature ismaintained at 37° C. by using a heating blanket. The surgical area isclipped and scrubbed. The animal is placed in right lateral recumbencyand initially placed on a ventilator with a peak inspiratory pressure of10-15 cm H₂O and respiratory rate 60-110 breaths/min. 100% O₂ isdelivered to the animals by the ventilator. The surgical site isscrubbed with surgical scrub and alcohol. An incision is made over therib cage at the 4^(th)-5^(th) intercostal space. The underlying musclesare dissected with care to avoid the lateral thoracic vein, to exposethe intercostal muscles. The chest cavity is entered through4^(th)-5^(th) intercostal space, and the incision expanded to allowvisualization of the heart. The pericardium is opened to expose theheart. A 6-0 silk suture with a taper needle is passed around the leftcoronary artery near its origin, which lies in contact with the leftmargin of the pulmonary cone, at about 1 mm from the insertion of theleft auricular appendage. The left coronary artery is occluded by tyingthe suture around the artery (“LCO”). Sham animals are treated the same,except that the suture is not tied. The incision is closed in threelayers. The rat is ventilated until able to ventilate on its own. Therats are extubated and allowed to recover on a heating pad. Animalsreceive buprenorphine (0.01-0.05 mg/kg SQ) for post operative analgesia.Once awake, they are returned to their cage. Animals are monitored dailyfor signs of infection or distress. Infected or moribund animals areeuthanized. Animals are weighed once a week.

7C. Efficacy Analysis

Six weeks after surgery, rats are scanned for signs of myocardialinfarction using ultrasound as described above. Only those animals withdecreased fractional shortening compared to sham rats are utilized inefficacy experiments. In all experiments, there are four groups:sham+vehicle, sham+compound, LCO+vehicle, and LCO+compound. At 7-12weeks post LCO, rats receive a bolus injection or are infused with testcompound. As in Example 6, five pre-dose M-Mode images are taken at 30second intervals prior to bolus injection or infusion of compound. Afterinjection, M-mode images are taken at 30 second intervals up to 10minutes, and thereafter every minute or at five minute intervals.Fractional shortening is determined from the M-mode images. Comparisonsbetween the pre-dose fractional shortening and post compound treatmentare performed by ANOVA and a post-hoc Student—Newman—Keuls with theStatView statistical program (SAS Institute). A p value %<0.05 isconsidered significant.

7D. Results

Compounds of the present invention show activity when tested by thismethod.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1. A method of potentiating cardiac myosin, which method comprisesadministering to a mammal in need thereof an amount of a compoundrepresented by Formula I effective to potentiate cardiac myosin

wherein: R¹ is alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl; R² is acetyl, acetylamino,acetylene, alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl,hydrogen, nitro or trifluoromethyl; and R³ is aryl, substituted aryl,heteroaryl or substituted heteroaryl; or a single stereoisomer, ormixture of stereoisomers, or a pharmaceutically acceptable salt thereof,wherein “substituted” alkyl, aryl, and heteroaryl refer respectively toalkyl, aryl, and heteroaryl wherein one to three hydrogen atoms arereplaced by a substituent independently selected from alkyl,fluoroalkyl, alkoxy, cyano, halogen, hydroxy, and nitro.
 2. The methodof claim 1 where R¹ is phenyl, substituted phenyl or pyridinyl.
 3. Themethod of claim 2 where R¹ is 3-chlorophenyl, 4-fluorophenyl,4-hydroxyphenyl, 3-methylphenyl, pyridin-2-yl or pyridin-3-yl.
 4. Themethod of claim 1 where R² is acetyl, acetylene, alkoxycarbonyl, cyano,halo, heteroaryl, hydrogen, nitro or trifluoromethyl.
 5. The method ofclaim 4 where R² is acetylene, cyano, ethoxycarbonyl, fluoro, hydrogen,nitro, oxazol-2-yl, pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl ortrifluoromethyl.
 6. The method of claim 1 where R³ is: phenyl,optionally substituted with lower alkoxy, lower alkyl, halo, hydroxy, ornitro; or furanyl, indolyl, isoxazolyl, pyrazolyl, pyridinyl orthiophen-2-yl, each of which is optionally substituted with lower alkyl,lower alkoxy, or halo.
 7. The method of claim 6 where R³ is3-hydroxyphenyl, 4-hydroxyphenyl, 4-fluorophenyl, isoxazol-5-yl,pyridin-3-yl, pyridin-4-yl, thiophen-2-yl or 5-methyl-thiophen-2-yl. 8.The method of claim 1 where R¹ is substituted phenyl and R² is nitro. 9.The method of claim 8 where R¹ is 4-fluorophenyl.
 10. The method ofclaim 1 where R¹ is heteroaryl and R² is fluoro.
 11. The method of claim10 where R¹ is pyridin-3-yl.
 12. The method of claim 1 where R¹ isheteroaryl and R² is nitro.
 13. The method of claim 12 where R¹ ispyridin-3-yl.
 14. The method of claim 13 where R³ is phenyl,4-fluorophenyl, 4-cyanophenyl, 4-methoxyphenyl or 4-methylphenyl. 15.The method of claim 1 where the compound is:N-[3-(4-fluoro-phenoxy)-(5-nitro-phenyl)]-isonicotinamide;4-fluoro-N-[3-(pyridin-3-yloxy)-phenyl]-benzamide;thiophene-2-carboxylic acid [3-(4-fluoro-phenoxy)-5-nitro-phenyl]-amide;isoxazole-5-carboxylic acid [3-(4-fluoro-phenoxy)-5-nitro-phenyl]-amide;N-[3-cyano-5-(pyridin-3-yloxy)-phenyl]-4-fluoro-benzamide;4-fluoro-N-[3-(pyridin-3-yloxy)-5-trifluoromethyl-phenyl]-benzamide;3-(4-fluoro-benzoylamino)-5-(pyridin-3-yloxy)-benzoic acid ethyl ester;5-methyl-thiophene-2-carboxylic acid[3-nitro-5-(pyridin-3-yloxy)phenyl]-amide;4-fluoro-N-[3-pyridin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;4-fluoro-N-[3-pyrazin-2-yl-5-(pyridin-3-yloxy)-phenyl]-benzamide;4-fluoro-N-[3-nitro-5-(pyridin-2-yloxy)-phenyl]-benzamide;N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-3-hydroxy-benzamide;N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-4-hydroxy-benzamide;N-[3-fluoro-5-(pyridin-3-yloxy)-phenyl]-nicotinamide; andisoxazole-5-carboxylic acid [3-fluoro-5-(pyridin-3-yloxy)-phenyl]-amide.